Variant Creutzfeldt-Jakob disease (vCJD) is a unique and highly distinctive clinicopathological and molecular phenotype of human prion disease associated with infection with bovine spongiform encephalopathy (BSE)–like prions. Here, we found that generation of this phenotype in transgenic mice required expression of human prion protein (PrP) with methionine 129. Expression of human PrP with valine 129 resulted in a distinct phenotype and, remarkably, persistence of a barrier to transmission of BSE-derived prions on subpassage. Polymorphic residue 129 of human PrP dictated propagation of distinct prion strains after BSE prion infection. Thus, primary and secondary human infection with BSE-derived prions may result in sporadic CJD-like or novel phenotypes in addition to vCJD, depending on the genotype of the prion source and the recipient.

Medical Research Council (MRC) Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.

* Present address: Department of Biochemistry and Molecular Biology and Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia.

To whom correspondence should be addressed. E-mail: j.collinge@prion.ucl.ac.uk

--------------------------------------------------------------------------------Distinct prion strains are associated with biochemically distinct forms of disease-related prion protein (PrPSc). Four PrPSc types have been observed in brain tissue from patients with distinct Creutzfeldt-Jakob disease (CJD) phenotypes: types 1 to 3 in classical (sporadic or iatrogenic) CJD and type 4 in vCJD (1–3). Polymorphism at residue 129 of human PrP (where either methionine or valine can be encoded) powerfully affects genetic susceptibility to human prion diseases (4–7) and appears to critically influence the propagation of these human PrPSc types. So far, types 1 and 4 PrPSc have been found only in humans homozygous for Met129; type 3 PrPSc is seen almost exclusively in individuals with at least one valine allele; and type 2 PrPSc has been commonly observed in all codon 129 genotypes (1–3). BSE and vCJD prion infection in transgenic mice expressing human PrP, but not mouse PrP (1, 8–10), indicates that codon 129 polymorphism determines the ability of human PrP to form type 4 PrPSc and to generate the neuropathological phenotype of vCJD. Challenge of transgenic mice expressing human PrP Met129 (129MM Tg35 and 129MM Tg45 mice) with BSE and vCJD prions (11) resulted in faithful propagation of type 4 PrPSc (10) (Figs. 1 and 2) accompanied by the key neuropathological hallmark of vCJD, the presence of abundant florid PrP plaques (10). However, transgenic mice expressing human PrP Val129 (129VV Tg152 mice) responded quite differently. Although these 129VV Tg152 mice lack a transmission barrier to classical forms of CJD, regardless of the codon 129 genotype of the inoculum (1, 8, 9), the primary challenge with vCJD prions was characterized by a substantial transmission barrier to infection (only 50% of inoculated mice were infected, compared with 100% of 129MM Tg35 and 129MM Tg45 mice) (Fig. 1; table S1). In addition, rather than type 4 PrPSc, vCJD-inoculated 129VV Tg152 mice propagated type 5 PrPSc (9), which shares the same predominance of the diglycosylated glycoform seen in type 4 PrPSc but is distinguished by proteinase K digestion products of greater molecular mass (Fig. 2A), which closely resemble those seen in human type 2 PrPSc (9). Type 5 PrPSc is associated with very weak diffuse PrP deposition in the brain (9), which contrasts markedly with the florid PrP plaques associated with the propagation of type 4 PrPSc in humans (12) or transgenic mice (10). Similar diffuse deposition of PrP is also observed in clinically affected BSE-inoculated 129VV Tg152 mice; however, type 5 PrPSc is undetectable in brain homogenate (9).

Fig. 1. Summary of transmissions of vCJD and BSE prions to transgenic mice. The total number of prion-affected mice (both clinical and subclinical) is reported for each inoculated group: 129MM Tg45 mice (black), 129MM Tg35 mice (gray), 129VV Tg152 mice (white). Animals were scored by clinical signs, immunoblotting, and/or immunohistochemistry. Primary transmission data have been published previously (9, 10). In transmissions that result in bifurcation of propagated PrPSc type, the number of samples positive for type 2 or type 4 PrPSc is reported as a proportion of the total number of samples examined by immunoblotting. (), The occurrence of subclinical prion infection only. [View Larger Version of this Image (16K GIF file)]

Fig. 2. Molecular strain typing of vCJD and BSE prion transmissions in transgenic mice. (A to D) Immunoblots of proteinase K–treated brain homogenates from variant and sporadic CJD (PRNP 129 MM genotype) and transgenic mice were analyzed by enhanced chemiluminescence with either monoclonal antibody 3F4 against PrP (A) or biotinylated monoclonal antibody ICSM 35 against PrP (B to D). The identity of the brain sample is designated above each lane with the type of PrPSc present in the sample designated below. Transmissions that result in the propagation of either type 2 or type 4 PrPSc. [View Larger Version of this Image (22K GIF file)]

To further evaluate the molecular and neuropathological phenotype of vCJD- or BSE-inoculated 129VV Tg152 mice, we performed a second passage in the same breed of mice. Primary transmission of prions from one species to another is associated with a species or transmission barrier that is largely or completely abrogated on second and subsequent passage in the second species as the prions adapt to the new host. Second passage then resembles within-species transmission with a high (typically 100%) attack rate and much shortened and more consistent incubation period. It was remarkable, however, that such adaptation did not occur on second passage of BSE or vCJD prions in 129VV Tg152 mice. Brain inocula derived from four clinically affected BSE-inoculated 129VV Tg152 mice failed to transmit clinical disease or asymptomatic prion infection to additional 129VV Tg152 mice (Figs. 1 and 3; table S2). However, two of these inocula produced clinical prion disease (Fig. 3; table S2) with abundant PrPSc accumulation (fig. S1) on inoculation of wild-type FVB mice with incubation periods that are not compatible with persistence of the original BSE inoculum [supporting online material (SOM) text]. The prion strain generated in BSE-inoculated 129VV Tg152 mice was thus infectious in wild-type FVB mice, but not in additional 129VV Tg152 mice.

Valine 129 is unique to human PrP, and the failure of BSE prions to adapt in 129VV Tg152 mice on second passage contrasts sharply with the marked adaptation of BSE prions in FVB mice (Fig. 3; table S2) or in other murine (13) or primate (14) hosts that encode methionine at the corresponding position of PrP. BSE prions also efficiently adapt on second passage in 129MM Tg35 transgenic mice. Primary transmission of BSE prions in 129MM Tg35 mice resulted in bifurcation of propagated strain type and produced either type 2 or 4 PrPSc (Figs. 1 and 2) and neuropathology consistent with human sporadic CJD or vCJD, respectively (10). These distinct molecular and neuropathological phenotypes consistently "breed true"with very high efficiency on second passage in additional 129MM Tg35 transgenic mice (15). These findings contrast sharply with the complete lack of prion transmission on second passage of the same BSE inocula in 129VV Tg152 mice, supporting the interpretation that human PrP Val129 severely restricts propagation of the BSE prion strain.

This conclusion was further reinforced by study of the parameters of second passage of vCJD prions. As seen with second passage of BSE prions, clinical disease was observed only in FVB, and not in 129VV Tg152, recipients. Brain inocula from clinically affected, type 5 PrPSc positive, primary vCJD-inoculated 129VV Tg152 mice produced clinical prion disease (Fig. 3; table S2) and PrPSc accumulation (fig. S1) on subpassage in FVB mice, but produced only subclinical infection (with PrPSc accumulation) in 7 out of 11 inoculated 129VV Tg152 mice (Figs. 1 and 3). Notably, in four of these, high-sensitivity methods (16) were required to detect PrPSc in brain homogenate (table S2). Type 5 PrPSc was faithfully propagated on second passage in 129VV Tg152 mice (Fig. 2A). In the three mice containing the highest levels of type 5 PrPSc, extensive spongiosis was also observed (Fig. 4), and in one of these, in contrast to the pathology seen on first passage, numerous PrP plaques were seen (Fig. 4). Type 4 PrPSc is invariably associated with prominent florid plaques in the cortex of human vCJD brain (12) and in vCJD- or BSE-prion inoculated 129MM Tg35 and Tg45 transgenic mice (10), whereas plaques associated with type 5 PrPSc were restricted to the corpus callosum and had a nonflorid morphology (Fig. 4). The lack of adaptation of vCJD prions on second passage in 129VV Tg152 mice contrasted sharply with the behavior of vCJD prions in wild-type FVB mice, where typical adaptation was observed on second passage with 100% clinical prion disease with abundant PrPSc accumulation (fig. S1) at markedly reduced incubation periods (Fig. 3; table S2).

Both BSE and vCJD prions failed to propagate efficiently on either primary or, remarkably, second passage, in 129VV Tg152 mice in sharp contrast to 129MM Tg mice or wild-type animals, and where detectable, infection was associated with a distinct PrPSc type and pathological phenotype. Thus, human PrP Val129 appears not to be a compatible substrate for propagation of the prion strain seen in vCJD. This interpretation was supported by the transmission properties of 129VV Tg152–passaged vCJD prions in 129MM Tg35 mice. Here, 14 out of 15 129MM Tg35 mice inoculated with isolates containing type 5 PrPSc showed PrPSc accumulation (Fig. 1; table S3), typically to much higher levels than seen in 129VV Tg152 mice receiving the same inocula. However, the PrPSc seen was not of the type 5 pattern but instead these transmissions mirrored the behavior of BSE prions in 129MM Tg35 mice (10), where, instead, either type 4 or type 2 PrPSc were seen (Figs. 1 and 2, B to D). Thus human PrPSc types 4 and 5 are restricted to propagating in mice expressing human PrP Met129 or Val129, respectively.

In conclusion, we have demonstrated that BSE and vCJD prion infection in transgenic mice can result in the propagation of distinct molecular and neuropathological phenotypes dependent on host PrP residue 129 and possibly other, as yet unidentified, disease modifying loci (10). These data predict a critical role for PRNP codon 129 in governing the thermodynamic permissibility of human PrPSc conformation that can be interpreted within a conformational selection model of prion transmission barriers (17–19) (SOM text) and suggest that there is no overlapping preferred conformation for Val129 and Met129 human PrP that can be generated as a result of exposure to the vCJD/BSE prion strain. Biophysical measurements suggest that this powerful effect of residue 129 on prion strain selection is likely to be mediated by means of its effect on the conformation of PrPSc or its precursors or on the kinetics of their formation, as it has no measurable effect on the folding, dynamics, or stability of the normal cellular prion protein PrPC (20).

Although caution must be exercised in extrapolating from animal models, even where, as here, faithful recapitulation of molecular and pathological phenotypes is possible, our findings argue that primary human BSE prion infection, as well as secondary infection with vCJD prions by iatrogenic routes, may not be restricted to a single disease phenotype. These data, together with the recent recognition of probable iatrogenic transmission of vCJD prions to recipients of blood (21, 22), including a PRNP codon 129 Met/Val heterozygous individual (22), reiterate the need to stratify all human prion disease patients by PrPSc type. This surveillance will facilitate rapid recognition of novel PrPSc types and of any change in relative frequencies of particular PrPSc subtypes in relation to either BSE exposure patterns or iatrogenic sources of vCJD prions.

There has been concern that the outbreak of mad cow disease (bovine spongiform encephalopathy, or BSE) in the United Kingdom would result in a large-scale spread of the infection to humans. Public worries, however, appear to have been allayed by the fading of the current epidemic (1) of the human form of mad cow disease, variant Creutzfeldt-Jacob disease (vCJD). Yet recent survey findings (2) and blood transfusion studies (3, 4) raise deep concerns among prion researchers that many more cases of undetected prion protein infection may underlie the overt epidemic. It is crucial to know whether such apparently dormant carriers are themselves infective and whether they are at risk of eventually developing clinical disease. The need for more extensive clinical surveys in the UK is a priority, but the urgency for these and other follow-up studies has been dampened by the disparate nature of recent findings, which are readily dismissed by some as "atypical" or of "uncertain significance." These reasons for inaction are rebutted by the experimental studies of Wadsworth et al. (5) reported on page 1793 of this issue. Using transgenic mice expressing the normal human prion protein, they show that an amino acid sequence variation (polymorphism) at position 129 of this protein drastically affects the infectivity and clinical consequences of BSE and vCJD infection.

The normal human prion protein expressed by brain neurons can undergo an aberrant change in conformation, resulting in misfolded forms that self-propagate. These aberrant prion proteins produce characteristic neurodegenerative changes in brain tissue resulting in a progressive and fatal encephalopathy (6, 7). This disease process occurs sporadically in humans: Each year one in a million deaths worldwide is attributed to the spontaneous development of Creutzfeldt-Jakob disease (CJD). Consequently, based on a life expectancy of 70 years, one in 15,000 people will die from CJD, with the likelihood that rather more than that number are infected but die from other causes. Thus, humans have always been exposed to CJD, but because the spread of the disease requires either the direct ingestion or injection of infected tissues, CJD has remained a sporadic disease confined to a few individuals. Historically, the great risk to our species from prion protein infection has come from cannibalism, as evidenced by the devastating kuru epidemic among the Fore tribe of Papua New Guinea. Evolution has provided some protection against this threat: Variations at critical amino acids in the normal human prion protein sequence limit susceptibility to infective propagation of aberrant forms of the prion protein (8). Notably, a polymorphism at position 129 of the normal human prion protein--either a valine (V) or a methionine (M)--provides some protection against kuru among 129MV heterozygous individuals. In contrast, 129MM homozygotes are particularly susceptible to prion infection (9).

The protective effect of a valine rather than a methionine at position 129 is evident in the current vCJD epidemic in the UK. This epidemic is a consequence of widespread infection of cattle with BSE from the early 1980s to 1996. During this period, hundreds of thousands of infected cattle entered the food chain (10). The consequent cross-species infection of humans with BSE resulted in the new variant form of prion encephalopathy called vCJD. This disease differs from sporadic human CJD in both brain tissue pathology and in the electrophoretic pattern that classifies each of the aberrant forms of prion protein. The UK vCJD epidemic, which now appears to be fading at 150 cases (1), has two striking features (see the figure). All of the affected individuals are 129MM homozygotes, and most are young, less than 30 years old. The tailing-off of this epidemic has been assumed by many to be the end of the vCJD threat, but to those involved in prion research it seems unlikely that infection would be confined to just one age group or to a single genotype. These fears are supported by recent findings.

A waning epidemic? Projected incidence in the UK of vCJD (deaths per 3 months), the human form of mad cow disease (1). A total of 150 people have been affected, all of whom carry one genotype (129MM), which is present in just 37% of the population (Inset). CREDIT: KATHARINE SUTLIFF/SCIENCE

In a UK survey of 12,700 surgically removed appendices, three stained positively for prion protein accumulation, indicating an unexpectedly high rate of infection, equivalent at a national level to thousands of infected individuals (2). Doubts as to the significance of the appendix survey findings have been answered by later studies of two recipients of blood transfusions from a donor who subsequently developed vCJD. The first recipient, who had a 129MM genotype, developed vCJD with typical clinical and histological changes 6 years after transfusion (3). But the critical findings came from autopsy of the second recipient, of genotype 129MV, who remained in good neurological health but died 5 years after the transfusion from a ruptured aortic aneurysm (4). Autopsy showed no evidence of brain involvement, but a pattern of prion protein accumulation was observed in lymphoid tissue similar to the diffuse deposition seen in the positive specimens in the appendix survey. The overall conclusion from these studies is that there are two levels of infection: one that results in overt vCJD, as in the genotype 129MM transfusion recipient, and another that results in a subclinical or dormant carrier state, as in the 129MV recipient.

These conclusions are strongly supported by the new study of Wadsworth et al. (5). These investigators analyzed transgenic mice expressing the 129MM or 129VV variant of the normal human prion protein for susceptibility to infection with BSE or vCJD. Exposure of 129MM mice to vCJD resulted in the consistent development of clinical disease, whereas 129VV mice were relatively resistant to infection. The infection that did occur in 129VV mice resulted in the atypical diffuse deposition of prion protein that was also observed in the human appendix and transfusion studies. Moreover, subpassage of brain tissue from infected 129VV mice resulted in typical vCJD infection among 129MM mouse recipients, but only in subclinical and atypical infection among 129VV mice.

These findings underscore the quandary faced by public health officials in the UK. Are the thousands of dormant carriers of vCJD indicated by the appendix survey at risk of developing clinical disease? Are they infective to others, or only to 129MM individuals, or not at all? Or are the survey findings just a manifestation of dormant sporadic CJD present in all populations? These questions need to be addressed with priority and urgency. The answers are vital to the future practice of blood transfusion, surgery, and dentistry in the UK and for health services in other countries. Progress is frustratingly slow. Essential follow-up studies and access to data are being hindered or even prevented by demands for patient anonymity (2) or by medico-legal concerns (4). Such reservations are out of proportion to the potential threats posed by a resurgence of vCJD infection in the UK. Progress will require political will. Meanwhile, prion researchers watch the inaction with dismay.

The author is at the Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, UK. E-mail: rwc1000@cam.ac.uk 10.1126/science.1106679Include this information when citing this paper.